Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA_A) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [³H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [³H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC₅₀ = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [³H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA_A receptors containing α6 and δ subunits. In contrast, [³H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC₅₀ ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [³H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [³H]EBOB binding with EC₅₀ ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC_50,1 ~ 16 nM and EC_50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABA_A receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA_A receptor populations.